Project Summary Mycobacteria produce two unusual polymethylated polysaccharides (PMPS), the 3-O-methylmannose polysaccharides (MMP) and the 6-O-methylglucose lipopolysaccharides (MGLP). Both polysaccharides are located in the cytoplasm where they have been postulated to regulate fatty acid and mycolic acid metabolism as a consequence of their ability to form stable 1:1 complexes with long-chain fatty acids and acyl-coenzyme A derivatives. These findings, however, have been derived from in vitro studies using purified polysaccharides and fatty acids and it is at present unclear whether they accurately reflect the physiological relevance of PMPS in intact mycobacterial cells. Studies initiated by our laboratory have begun to shed light on the nature of the enzymes involved in MGLP biosynthesis and to yield the first recombinant strains defective in various aspects of MGLP biosynthesis. Preliminary analyses of these strains point to an important role of MGLP in the adaptation of Mycobacterium tuberculosis and Mycobacterium smegmatis to thermal stress. Consistent with this observation, earlier work by Dr. Ballou and associates revealed that a M. smegmatis strain partially deficient in PMPS production displayed altered levels of unsaturated fatty acids. Taken together, these results are suggestive of the existence of a rather unique mechanism used by mycobacteria to control fatty acid metabolism in response to stress. Whether MGLP are essential for mycobacterial growth is currently unknown as none of the mutants generated so far were totally defective in MGLP biosynthesis and their growth was only tested under a limited number of laboratory conditions. Likewise, the precise function of MGLP in the fatty metabolism of intact cells remains essentially undefined since mutant characterization thus far has been restricted to the analysis of steady-state levels of fatty acids under conditions where MGLP may not be required for optimal growth, and has neither thoroughly analyzed longer acyl chains such as mycolic acids, addressed de novo synthesis of fatty acids and mycolic acids, or analyzed lipid contents. We propose under Aim 1 to knock-out or knock-down in M. tuberculosis a number of MGLP biosynthetic genes that we identified so as to determine whether MGLP are required for growth under a variety of conditions thought to mimic the physical environments encountered by M. tuberculosis during host infection. Under Aim 2, we will use these mutants as well as other recombinant strains presenting various quantitative or qualitative defects in MGLP synthesis that we will generate to shed light on the physiological function(s) of these polysaccharides with a particular focus on fatty acid, mycolic acid and lipid metabolism. In addition to advancing our understanding of a novel and potentially critical physiological process of mycobacteria, our proposed studies may prove useful in designing innovative and Mycobacterium-specific therapeutic strategies for the treatment of tuberculosis and other mycobacterial infections.